Photocontrolled dynamic covalent linkers for polymer networks

ABSTRACT

Reversibly crosslinkable polymeric networks, including reversibly crosslinkable hydrogel networks are provided. Also provided are methods of making the polymeric networks and methods of using the hydrogel networks in tissue engineering applications. The reversibly crosslinkable polymeric networks are composed of polymer chains that are covalently crosslinked by azobenzene boronic ester bonds that can be reversibly formed and broken by exposing the polymeric networks to different wavelengths of light.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 17/055,777 filed Nov. 16, 2020, which claimspriority as a National Stage Application of International Applicationnumber PCT/US19/32881 filed May 17, 2019, which claims the prioritybenefit of U.S. patent application Ser. No. 62/673,312, filed May 18,2018, the contents of all of which are incorporated by reference hereinin their entirety.

BACKGROUND

Polymer networks crosslinked with dynamic bonds can be self-healing,adaptive, and recyclable. The conditions under which these propertiesare observed depend on the stability and lifetime of the dynamic bonds.By tailoring crosslink stability and reactivity, macroscopic propertiescan be programmed at the molecular level. Furthermore, if changes incrosslink density or dynamics occur in response to a stimulus, thesematerials exhibit tunable macroscopic properties. External stimuli suchas pH, temperature, and magnetic field have been employed to reversiblytune the properties of dynamic polymer networks.

As soft materials with mechanics and water content that approximatethose of tissues, hydrogels benefit from the introduction of reversible,externally controlled properties. While traditional stimuli such as pHor temperature present limitations on biocompatibility, light(particularly in the visible to near-infrared (IR) range) represents anideal stimulus. Light can be applied externally with precise spatial andtemporal control, at controlled wavelengths and fluxes. However, themajority of photocontrolled hydrogels rely on irreversible photochemicalreactions, such as photoinitiated radical polymerization for stiffeningor o-nitrobenzyl cleavage for softening. In addition to theirirreversibility, these reactions require exogenous reagents or releasebyproducts into the hydrogel matrix.

Reversible light-responsive hydrogels are comparatively rare due to thelimited number of photoreversible reactions that can be coupled to achange in hydrogel stiffness. Covalently linked hydrogels based onphotoreversible [2+2] cycloadditions display reversible stiffening andsoftening at low concentrations. While recent work has achieved thecycloaddition with visible light, the reverse reaction invariablyrequires ultraviolet (UV) irradiation. As an alternative tophotoreversible reactions, many researchers have turned to thephotoswitch azobenzene, which undergoes reversible E/Z isomerization inresponse to two different wavelengths of light. Rosales and coworkersenchained azobenzene in an elastic network and observed small butreproducible changes in stiffness. (See, Rosales et al.,Biomacromolecules. 2015, 16, 798.) Notably, the above systems are notdynamic in the absence of light; these elastic networks store, ratherthan dissipate, energy from applied strain. To achieve a sol-geltransition in a stress-relaxing network, Harada and coworkers designed asupramolecular hydrogel based on cyclodextrin/azobenzene complexes thathas been leveraged in multiple contexts. (See, Tamesue et al., Angew.Chem. Int. Ed. 2010, 49, 7461; Tomatsu, et al., J. Am. Chem. Soc. 2006,128, 2226; and Yamaguchi et al., Nat. Commun. 2012.)

SUMMARY

Reversibly photo-crosslinkable polymeric networks, including reversiblyphoto-crosslinkable hydrogel networks, are provided. Also provided arecell culture scaffolds and hybrid hydrogel materials made from thephoto-crosslinkable polymeric networks, as well as methods for using thecell culture scaffolds in tissue engineering applications.

One embodiment of a hydrogel includes a crosslinked polymer networkcomprising covalent azobenzene boronic ester crosslinks between theorganic polymer backbone chains.

One embodiment of a cell culture scaffold includes: a hydrogelcomprising a crosslinked polymer network comprising covalent azobenzeneboronic ester crosslinks between the organic polymer backbone chains;and biological cells seeded on the hydrogel or encapsulated in thehydrogel.

One embodiment of a hybrid hydrogel material includes: a hydrogelcomprising a crosslinked polymer network comprising covalent azobenzeneboronic ester crosslinks between the organic polymer backbone chains;and a fiber-forming biomaterial, wherein the hydrogel and thefiber-forming biomaterial form an interpenetrating network.

Another embodiment of a cell culture scaffold includes: a hybridhydrogel material and biological cells seeded on the hybrid hydrogelmaterial or encapsulated in the hybrid hydrogel material. The hybridhydrogel material includes: a hydrogel comprising a crosslinked polymernetwork comprising covalent azobenzene boronic ester crosslinks betweenthe organic polymer backbone chains; and a fiber-forming biomaterial,wherein the hydrogel and the fiber-forming biomaterial form aninterpenetrating network.

The polymer networks, including the hydrogels can be formed from areversibly crosslinkable composition that includes: a first moleculecomprising at least two terminal azobenzene boronic acid groups; and asecond molecule comprising at least two terminal diol groups, providedthat at least one of the first and second molecules is a polymer, andfurther provided that, if the first molecule has only two terminalazobenzene boronic acid groups, then the second molecule comprises atleast three terminal diol groups, and if the second molecule has onlytwo terminal diol groups, the first molecule has at least three terminalazobenzene boronic acid groups; wherein the first molecule ischaracterized in that its azobenzene groups undergo a reversible E to Zisomerization upon irradiation with ultraviolet light, visible light, orinfrared light, and its boronic acid groups react with the terminal diolgroups of the second polymer to form a polymer network comprisingcovalent azobenzene boronic ester bonds.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention will hereafter be describedwith reference to the accompanying drawings.

FIG. 1A shows an illustrative example of an azobenzene boronic acid endgroup. FIG. 1B shows an illustrative example of a diol group-containingend group. FIG. 1C shows the chemical structure (left) and a schematicrepresentation (right) of one example of a branched polymer having diolend groups. FIG. 1D shows the chemical structure (left) and a schematicrepresentation (right) of one example of a branched polymer havingazobenzene boronic acid end groups. FIG. 1E shows crosslinks between afirst polymer and a second polymer. FIG. 1F shows molecular crosslinksbetween polymer chains of a first polymer.

FIG. 2 shows the reversible crosslinking of a polymer network usingazobenzene boronic acid functionalities.

FIG. 3 depicts structural modifications to an azobenzene boronic acidcrosslinking group. The black dot represents the attachment point of theend groups to the polymer backbone.

FIG. 4 depicts a diol-modified hyaluronic acid and a telechelicazobenzene boronic acid linker that can be used to make a HA-derivedphotoresponsive gel.

FIG. 5 depicts a small-molecule model system for studying the relativerates and equilibrium constants for reversible esterification of ano-azobenzene boronic acid.

FIG. 6A depicts esterification of 400 μM mixture of (E)- and (Z)-1 with40 mM of pinacol in 1:1 ACN:H₂O. FIG. 6B depicts linear fit of data fromFIG. 6A to determine apparent rates of esterification and hydrolysis ofE and Z isomers. FIG. 6C depicts hydrolysis of 400 M mixture of (E)- and(Z)-2 in 1:1 ACN:H₂O. FIG. 6D depicts linear fit of data from FIG. 6C toconfirm apparent rates of hydrolysis for E and Z isomers.

FIG. 7 depicts the structure of azobenzene- and diol-terminatedpoly(ethylene glycol) polymers P1-P4.

FIG. 8 shows the reversible sol-gel transition of a mixture of P1 and P2(1:1, 10 w/v % in PBS, pH 7.5).

FIGS. 9A-9C depict the representative photorheological characterizationof the hydrogels of Example 1. FIG. 9A shows the UV-induced gelationprofile of P1 and P2 (1:1, 10 w/v % in PBS, pH 7.5, 10% strain, 25rad/s). FIG. 9B depicts the photocontrolled cycling of hydrogelviscoelasticity (10% strain, 25 rad/s). UV light induces gelation, whichis reversed with blue light. UV light is required to re-initiatestiffening of the gel. FIG. 9C shows the dynamic frequency sweepmeasurements as a function of irradiation (10% strain). Inset: stressrelaxation of the hydrogel (gelled for 60 minutes) after applying 10%strain.

FIG. 10A depicts the photocontrolled cycling of hydrogel viscoelasticity(10% strain, 25 rad/s) of P1 and P2-F₂ (1:1, 10 w/v % in PBS, pH 7.5).Green light induces gelation, and blue light induces softening. FIG. 10Bshows the stress-relaxation of the gel as a function of irradiationtime.

FIG. 11A depicts the structure of a diol-grafted alginate. Thediol-grafted alginate was combined with a telechelic bis-azobenzeneboronic acid linker, and the resulting alginate gels werephotoresponsive (FIG. 11B) and exhibited slower stress relaxation thanP1 gels with the same linker (FIG. 11C).

FIG. 12 depicts the photoresponse of hybrid P1/P2-F₂/collageninterpenetrating network.

FIG. 13 depicts preliminary data for a gel/liver extracellular matrix(ECM) hybrid interpenetrating network with oscillatory shear rheology.

FIG. 14 shows a synthetic route of small molecules 1 and 2 for kineticstudies and compound SI-2 and SI-5 for synthesis of control polymers P2and P3 in Example 1.

DETAILED DESCRIPTION

Reversibly photo-crosslinkable polymeric networks, including reversiblyphoto-crosslinkable hydrogel networks, are provided. Also provided aremethods of making the polymeric networks and methods of using thehydrogel networks in tissue engineering applications.

The hydrogel networks are viscoelastic; like natural tissues, they havesolid-like and liquid-like characteristics. Photocontrolled dynamiccrosslinkers in hydrogel networks allow for rational spatiotemporaltuning of the mechanical properties and biochemical content of thematerials, rendering the materials useful as synthetic biomaterials. Therational tuning of the gel viscoelasticity with light enables thesynthesis of gels having a range of mechanical properties, includingphysiologically relevant mechanical properties. Because the viscoelasticproperties of the gels can be controlled both temporally and spatially,the gels have the ability to mimic biological tissues, such as the ECM,which have heterogenous mechanical properties that can change over time.

The reversibly crosslinkable polymeric networks are composed of polymerchains that are covalently crosslinked by azobenzene boronic estercrosslinks. The azobenzene boronic ester crosslinks are dynamic covalentcrosslinks that can be reversibly formed and broken by exposing thepolymeric networks to different wavelengths of UV, visible, and/or IRlight. As a result, the density of crosslinks and the viscoelasticproperties of the polymeric networks can be tailored by exposure light.The stiffness (G′, storage modulus) of the crosslinked gels increases asa function of irradiation time and, therefore, the viscoelasticproperties of the gels can be externally tailored by controlling theirradiation time. Notably, this external tuning of the hydrogelstiffness can be achieved independent of stress relaxation. This issignificant for tissue engineering applications because substratestiffness and substrate stress relaxation can independently control cellfate.

The polymeric networks and gels can be formed from compositions thatinclude a mixture of a first molecule having at least two reactiveterminal azobenzene boronic acid groups—that is, terminal boronicacid-substituted azobenzene groups—and a second molecule having at leasttwo reactive terminal diol groups, wherein at least one of the twomolecules has at least three of its respective reactive terminal groups.

The external control of the mechanical properties of the gels isprovided by the small-molecule azobenzene boronic acid and —OH endgroups, which can be installed on any synthetic or biological moleculeor polymer of interest. An illustrative example of an azobenzene boronicacid group is shown in FIG. 1A and an illustrative example of a diolgroup-containing end group is shown in FIG. 1B.

The molecules bearing the azobenzene boronic acid and diol end groupscan be small molecules or polymers. As used herein, the term polymersencompasses oligomers which are comprised of only a relatively fewrepeat units, for example, 3-5 repeat units. In some embodiments, atleast one of the two molecules is a polymer. For example, the moleculehaving at least three reactive terminal groups may be a branchedpolymer. In some embodiments, both the first and the second moleculesare branched polymers. The chemical structure (left) and a schematicrepresentation (right) of one example of a branched polymer having diolend groups is shown in FIG. 1C, and the chemical structure (left) and aschematic representation (right) of one example of a branched polymerhaving azobenzene boronic acid end groups is shown in FIG. 1D. Forpurposes of illustration, both polymers are polyethylene glycol polymersin FIGS. 1C and 1D. However, other polymers can be used, as discussed inmore detail below, and the polymer that forms the backbone of the diolgroup-bearing polymer need not be the same polymer that forms thebackbone of the azobenzene boronic acid group-bearing polymer.

Crosslinks are formed between polymer chains in the composition when theboronic acid substituents react with the diol groups. The crosslinks maybe between the first molecule and the second molecule if both moleculesare polymers; this is illustrated schematically in FIG. 1E.Alternatively, if only one of the first and second molecules is apolymer, the crosslinks can be formed between polymer chains of thatpolymer, as shown schematically in FIG. 1F. In these systems, theequilibrium of the reaction between the azobenzene boronic acid groupsand diol groups to form the boronic ester is controlled by theconfiguration of the azobenzene groups, which act as photoswitches forthe reaction.

Although the azobenzene-group-containing molecules may have only twoterminal azobenzene boronic acid groups, they may also have more thantwo such groups. For example, the molecules may be tri- or higherfunctional small molecules or branched polymers having at least three orat least four azobenzene boronic acid groups. Similarly, thediol-group-containing molecules may have only two terminal diol groups,but they may also have more than two such groups. For example, thesemolecules also may be tri- or higher functional small molecules orbranched polymers having at least three or at least four terminal diolgroups. A variety of molecules can be used in the compositions includingsynthetic and naturally occurring polymers, provided they can befunctionalized with azobenzene boronic acid groups or diol groups.

The reversible crosslinking of a polymer network using azobenzeneboronic acid functionalities is illustrated in FIG. 2. When theazobenzene group is in its E form (right panel), the equilibrium in anaqueous dispersion favors the boronic acid and crosslinking isdisfavored. However, when the azobenzene group is in its Z form (leftpanel), boronic ester crosslink formation is favored. The isomerizationfrom the E form to the Z form of the azobenzene group can be induced byexposing the molecules (e.g., polymers) to UV light, visible light, orIR light, including near-IR (hv₂), while the reverse isomerization fromthe Z form to the E form can be induced by exposing the resultingnetwork to a different wavelength of visible and/or UV light, includingblue light (hv₁). As a result, upon irradiating a mixture of moleculesfunctionalized with azobenzene-boronic acids and diols with twodifferent wavelengths of light, crosslinks in the covalent network canbe formed and re-broken. These cycles can be performed repeatedly.

In some embodiments of the compositions, the polymer network that formsupon crosslinking is a highly crosslinked gel, such as a hydrogel. Ifhydrogel formation is desired, the polymers used to form the crosslinkednetwork should be hydrogel-forming polymers, such as poly(ethylene)glycol polymers. Other hydrogel forming polymers include naturalpolymers, such as glycosamineglycans, including hyaluronic acid,polysaccharides, such as alginate, gelatin, and dextran, and syntheticpolymers, such as polyvinylalcohol, polyacrylamides, polyesters, andpolyacrylates. These and other polymers can be modified to incorporatediol groups using known chemistries. By way of illustration, aring-opening reaction of δ-gluconolactone with the polymer can be usedto form a water-soluble, unhindered penta-ol-grafted polymer, which canbe reacted with an azobenzene boronic acid crosslinker to form a gel.The modification of alginate with diol-bearing end groups is illustratedin Example 2.

In addition, it may be desirable to use polymers having three or moreterminal azobenzene boronic acid groups. The hydrogels may be formedfrom a starting composition that is a hydrosol, such that sequentialexposure of the composition to two different wavelengths of lightinduces a reversible sol-gel transition. However, the initialcomposition may also undergo some initial crosslinking to form ahydrogel, in which case, the sequential exposure of the composition totwo different wavelengths of light induces a stiffening and thensoftening of the hydrogel.

Mechanical properties of the gels, such as stiffness and stability, canbe adjusted by chemical modifications to the azobenzene groups. Forexample, an azobenzene ring having fluoride substitutes, such aso-difluoroazobenzene, can be used to provide a higher binding affinitybetween the azobenzene boronic acid and the diols. Also, crosslinkingcan be promoted and, therefore, gel stiffness increased by incorporatingelectron-withdrawing groups on the azobenzene group. Electronwithdrawing groups, such as CF₃, NO₂, SO₃ ⁻, and NMe₃ ⁺, para to theboronic ester group will favor boronic ester crosslink formation, due tothe increased Lewis acidity of the boronic acid group. Examples ofazobenzene boronic acid end groups are shown in FIG. 3.

One way to extend the lifetime of the dynamic covalent hydrogels todays-weeks, while maintaining stress relaxation, is to incorporate asmall percentage of permanent crosslinks in the gels. This percentagecan be a percentage above the gel point, as determined by the Carothersor Flory-Stockmayer approximations. In various embodiments of the gels,the mole percent of monomers that form permanent crosslinks can be inthe range of, for example, 0.1% to 20%, including in the range of 1% to10%. However, higher or lower mole percent ranges can be used.

The resulting hydrogels are viscoelastic but do not relax stress fullyor dissolve. Azide-alkyne, thiol-ene, and/or amide functionalities areexamples of functionalities that can be incorporated into one or both ofthe molecules/polymers that make up the gels to provide permanentcrosslinking.

The properties of the gels can also be modulated by the relative amountof the two molecules (e.g., polymers) in the compositions, which can bevaried over a relatively wide range. By tuning the ratio of the twomolecules, the gel stability and/or photoresponse can be modulated. Byway of illustration, the weight ratio of the first molecule to thesecond molecule in some embodiments of the compositions is in the rangefrom about 1:2 to about 2:1, including within the range from about 1:1.5to 1.5:1. However, weight ratios outside of this range can also be used.

As illustrated in the Example, the hydrogels can be viscoelastic,exhibiting stress relaxation on the order of seconds (e.g., half-life,t_(1/2)≤100 seconds), and the stiffness can be tuned independently ofthe crossover frequency. Some embodiments of the hydrogels have amaximum storage modulus (G′) of at least 200 Pa, where the storagemodulus of a hydrogel can be measured as described in the Example 1. Byway of illustration, some embodiments of the hydrogels have a G′ in therange from 1 Pa to 3500 Pa. Moreover, because different functionalgroups are used for crosslinking and photoexcitation, the photophysicsof the system can be readily modulated without compromising reactivity.

Polymer networks and gels having spatially heterogeneous mechanicalproperties can be formed by irradiating different regions of thenetworks and gels for different time durations, different radiationintensities, and/or different wavelengths of radiation. In this way, thehydrogels can be designed to mimic various synthetic and naturallyoccurring materials, such as biological organs which are composed ofmultiple types of tissue microenvironments having distinct stiffnessproperties. The stiffness patterns and gradients can be erased and/ormodified by one or more additional light patterning steps. This spatialand temporal control of the viscoelastic properties of the networks andgels can be achieved, for example, by irradiating the networks and gelsthrough a photomask or using confocal microscopy. Using thesetechniques, relatively stiff and relatively soft regions and/orstiffness gradients can be patterned into a gel in the presence ofbiological cells. The G′_(max) of the relatively stiff and relativelysoft regions can differ by a factor of two or more. For example, the tworegions can have G′_(max) values that differ by a factor of at least 10,a factor of at least 100, or a factor of at least 1000. In addition, oneor more areas of the gel can remain non-irradiated, and thesenon-irradiated areas will remain in the sol state and be removed byrinsing.

The ability to provide reversible photocontrolled spatially andtemporally tunable viscoelastic properties is significant for tissueengineering applications because different cellular microenvironmentscan present spatiotemporally heterogeneous cues, which can affectspreading, migration, proliferation, organ function, and stem celldifferentiation. By tuning the properties of hydrogels used as cellculture substrates, cyclic and complex temporal cues in biology (e.g.ovulation, hormone sensing and release, stem cell differentiation,age-related fibrosis, and tumor metastasis) can be mimicked.

Because hydrogels with spatially and temporally tunable viscoelasticproperties can be made using the present methods, the hydrogels can beused for a variety of applications. For example, hydrogels havingmechanical properties and water contents that approximate those ofliving biological tissues can be used as cell culturing scaffolds fortissue engineering applications, provided the polymers are bio- andcytocompatible. By way of illustration, gels having G′ values in therange from 0.1 to 1 kPa can be formed to mimic brain tissue and nervoustissue; gels having G′ values in the range from 1 to 10 kPa can beformed to mimic liver, fat, breast gland, relaxed muscle, and tendontissues; gels having G′ values in the range from 10² to 10³ kPa can beformed to mimic dermis, contracted muscle, and cartilage tissues; andgels having G′ values in the range from 10⁵ to 10′ kPa can be formed tomimic bone.

The hydrogels can be used as tissue growth scaffolds by seeding thescaffolds with living biological cells or by photogelation of thecompositions that also include the biological cells. Tissue can be grownby culturing the seeded scaffolds in a cell growth culture medium. Humanmesenchymal stem cells, hematopoetic stem cells, embryonic stem cells,pluripotent stem cells, osteoblasts, chondrocytes, fibroblasts,endothelial cells, and myocytes are examples of the types of cells withwhich the scaffolds can be seeded.

Modified hyaluronic acid (HA) is an example of a diol group-bearingpolymer that can be used to form a hydrogel that mimics ECM. HA isanimal derived but available as reproducible, highly purified(growth-factor-free) samples in a variety of molecular weights. HA is astructural component in ECM and regulator of cell behavior, and is usedas the key polymeric component for many semi-synthetic biomaterials. HAcan be modified with diol end groups by grafting HA withδ-gluconolactone (FIG. 4, top panel). An HA-based gel can then be formedby photocrosslinking the modified HA with a water-soluble,bis-azobenzene boronic acid linker, such as L-1c (FIG. 4, bottom panel).By way of illustration only, HA polymers having molecular weights in therange from 5 kDa to 5000 kDa can be used.

In other embodiments of an ECM mimicking material, a reversiblyphotocrosslinkable gel is combined with a fiber-forming biomaterial,such as collagen, fibronectin, or ECM itself to create materials thatpreserve the fibrillar architecture of ECM, while enablingspatiotemporal control. The fiber-forming biomaterials are able to formfibrillar supramolecular aggregates. These materials, which compriseinterpenetrating networks of a fiber-forming biomaterial (e.g.,decellularized ECM or fiber-forming ECM components) and a synthetichydrogel of a type described herein as different phases, combine theadvantages of photocontrol with the biochemical signals and fibrousmorphology of ECM. This fibrillar architecture is important for celladhesion and migration. The interpenetrating networks have tunablemechanics attributable to the reversible crosslinking properties of thepolymers in the gel. The ratios of diol end group-bearing polymers,azobenzene boronic acid crosslinker, and fiber-forming components can beadjusted to tailor the mechanical properties and photoresponse of thematerials. The interpenetrating networks can be prepared by pre-mixingthe diol end group-bearing polymers, azobenzene boronic acidcrosslinker, and fiber-forming components and incubating the resultingcomposite at, for example, 37° C. Alternatively, the film-formingcomponent can be molded at, for example, 37° C. and then combined with asolution of the gel-forming components.

An example of an interpenetrating network formed from collagen and areversibly photocrosslinkable diol-functionalized PEG polymer isdescribed in Example 3. An interpenetrating network of collagen andother polymers, such as a diol-functionalized HA polymer, could also besynthesized. An example of an interpenetrating network formed fromdecellularized ECM and a reversibly crosslinkable diol-functionalizedPEG polymer is described in Example 4. An interpenetrating network ofdecellularized ECM and other polymers, such as a diol-functionalized HApolymer, could also be synthesized. The interpenetrating networks can beused for a variety of applications.

As cell culturing materials and growth scaffolds, the gels andinterpenetrating networks can be used to study and control a variety ofcell behaviors as a function of substrate stiffness and/or stressrelaxation. For example, the gels and interpenetrating networks can beused to study or control cell spreading as a function of stiffness forcells that are seeded on or encapsulated within the gels andinterpenetrating networks. The gels and interpenetrating networks canalso be used to study or control the effects of temporally changingstiffness (soft to stiff or vice versa) on cell fate. For the syntheticECM-based gels and interpenetrating networks, such studies haverelevance to developmental and disease processes, which arecharacterized by increasingly stiff tissues.

EXAMPLES Example 1: Poly(ethylene glycol)-Based Hydrogels

In this example, the development of a platform to reversibly tune themechanical properties of dynamic hydrogels that use photoswitches tocontrol the reactivity of dynamic covalent crosslinks is reported.Small-molecule studies suggest that the conformation of the azobenzeneboronic acid determines the equilibrium constant for condensation withdiol, with an increase in K_(eq) for the Z isomer. The increase in theequilibrium constant generates a higher crosslink density in thehydrogel network, resulting in stiffening. Because of the dynamic natureof the boronic ester crosslink, these hydrogels are viscoelastic andstress-relaxing, and have stiffness that can be tuned independently ofcrosslink exchange rates. This approach can be generalized to ano-difluoroazobenzene with superior photophysical properties, enablingmechanical control of the hydrogel solely with visible light.

A small-molecule model compound, azobenzene 1, was first designed, inwhich the boronic acid was positioned ortho to the azo group (FIG. 5).Irradiation of (E)-1 with 365-nm light provided a 88:12 mixture of Z andE isomers. This mixture was subjected to excess pinacol inacetonitrile-water (1:1 v/v, 25° C.) to determine the apparent rates andequilibrium constants of boronic acid (1) esterification and boronicester (2) hydrolysis for each isomer. While other 1,2- and 1,3-diolswere hydrolyzed too quickly to be studied, the unusually slow rate ofpinacol hydrolysis allowed the E and Z isomers to be resolved and theirreaction kinetics to be monitored by high-performance liquidchromatography (HPLC; see Supporting Information (SI) for details).

This initial experiment revealed a difference in the reactivity of (E)-1and (Z)-1. After 8 hours, the reactions had reached equilibrium, with39% conversion of (Z)-1 to (Z)-2 and only 9% conversion of (E)-1 to(E)-2 (FIG. 6A). Using a reversible pseudo-first-order kinetic model, itwas determined that the esterification of (Z)-1 was 2.1 times fasterthan the esterification of (E)-1 (k₁) (FIG. 6B, Table 1). The rates ofhydrolysis could also be extracted from this model: (E)-2 underwenthydrolysis 2.0 times faster than (Z)-2 did (k⁻¹). These apparent rateconstants for hydrolysis were verified by hydrolyzing a mixture of (E)-2and (Z)-2 under irreversible pseudo-first-order conditions (FIGS. 6C and6D). Taken together, the apparent equilibrium to form boronic ester fromboronic acid and pinacol is 4.3 times more favorable for the Z isomerrelative to the E isomer. While convenient for small-molecule kineticstudies, the rate of pinacol ester formation is too slow to be practicalfor gelation. Thus, a less sterically hindered diol was used forhydrogel studies.

TABLE 1 Apparent rate and equilibrium constants for the small-moleculemodel study. Data are the average of three experiments. ConfigurationK_(eq) ^([a]) k₁ (s⁻¹) ^([a]) k⁻¹ (s⁻¹) ^([a]) k⁻¹ (s⁻¹) ^([b]) E 0.090± 0.016 2.56 ± 0.28 × 10⁻⁵ 2.76 ± 0.32 × 10⁻⁴ 2.83 ± 0.06 × 10⁻⁴ Z  0.39± 0.024 5.39 ± 0.45 × 10⁻⁵ 1.39 ± 0.20 × 10⁻⁴ 1.45 ± 0.10 × 10⁻⁴ Z/E 4.32.11 0.504 0.512 ^([a]) Apparent equilibrium constants and rateconstants obtained from the reversible esterification experiment. ^([b])Apparent rate constants obtained from the irreversible hydrolysisexperiments.

Hydrogel Synthesis and Rheological Characterization

With these small-molecule data in hand, the next step was to translatethe molecular design to photoswitchable networks. A pair of branchedpolymers, P1 and P2, was prepared with complementary diol and boronicacid functionalities (FIG. 7). The diol-terminated polymer (P1) wassynthesized by ring opening glucono-δ-lactone with amine-terminated4-arm poly(ethylene glycol) (PEG, Mw=5 kDa) according to a literatureprocedure. (See, Yesilyurt et al., Adv. Mater. 2017, 29; and Yesilyurt,et al., Adv. Mater. 2016, 28, 86.) The boronic acid polymer (P2) wassynthesized by coupling the same PEG with the carboxylic acid derivativeof compound 1 using carbodiimide coupling chemistry (see SI fordetails). Control polymers P3 and P4 were synthesized in analogy to P2to evaluate the role of the ortho-boronic acid.

To qualitatively investigate the effect of irradiation on the boronicester hydrogel, P1 and P2 were mixed in a 1:1 ratio of 0.1 Mphosphate-buffered saline (PBS) at pH 7.5 (10 w/v %). Prior toirradiation, the mixture was a sol, according to the flow-inversionmethod. Irradiation with a 365 nm flashlight for 10 minutes inducedpartial E to Z isomerization of the azobenzene photoswitch and led togelation. Irradiating this gel for 30 seconds with blue LEDs (470 nm)caused Z to E isomerization and returned the mixture to the sol state.The sol-gel cycles could be repeated multiple times by sequentialirradiation with 365 and 470 nm light (FIG. 8).

In contrast, P1 and control polymer P3 (a para-boronic acid) formed agel without irradiation, and this gel was not photoresponsive. Thisobservation indicates that proximity to the azo group, rather than aninductive/resonance or rigidity effect, was responsible for thephotoresponse. The combination of P1 and P4, lacking a boronic acid,formed a sol regardless of irradiation, providing evidence that theboronic ester was the crosslink. (See SI for rheologicalcharacterization of the control gels.)

To quantitatively assess the photoresponsive bulk mechanical propertiesof the hydrogel, photo-oscillatory rheology was performed at constantstrain and frequency within the linear viscoelastic regime. Uponconstant irradiation of mixtures of P1 and P2 (1:1, 10 w/v % in PBS, pH7.5) with 365-nm LED light, the storage (G′) and loss moduli (G″), whichrepresent the elastic and viscous characteristics of the hydrogel,respectively, increased by over an order of magnitude. The maximumstorage modulus (220 Pa) was achieved after approximately 3 hours ofirradiation (FIG. 9A). Importantly, it could be quantitativelydemonstrated that this change in mechanical properties was reversible byperforming photorheology with alternating 365- and 470-nm light (FIG.9B). After stiffening the gel with 365-nm light for 2 hours, irradiationwith 470-nm light for 2 minutes returned the network to its originalstate. Gelation could be repeated by irradiation with 365-nm light. Incontrast to strategies based on photocleavage or photoinitiatedpolymerization, water was the only byproduct and required an exogenousreagent.

Interestingly, this system stiffened in the Z conformation and softenedin the E conformation. While the measured rate and equilibrium constantscannot be directly correlated for the small-molecule model system(Table 1) to the viscoelastic behavior of the P1/P2 hydrogel due to thechange in diol, it is believed that the Z azobenzene boronic acidexperienced more favorable equilibrium towards the boronic estercompared to the E isomer. Since the boronic ester was the elasticallyeffective crosslink, a higher equilibrium corresponded to highercrosslink density and thus a stiffer gel.

The viscoelastic properties of the hydrogel system were nextcharacterized as a function of irradiation. Networks formed fromirreversible covalent bonds are elastic, and exhibit frequencyindependent moduli because the crosslinks are fixed. Dynamicallycrosslinked networks have time-dependent properties. At fast timescales, the oscillation occurs faster than bonds can rearrange, thus thenetworks behave as gels. At slow time scales, crosslinks can exchangefaster than the oscillation, and the networks behave as liquids. Thecrossover frequency (ω_(c)) at which G′ and G″ are equal corresponds tothe oscillation frequency at which the viscoelastic material transitionsfrom solid to liquid.

Frequency sweeps were performed on mixtures of P1 and P2 (1:1, 10 w/v %in PBS, pH 7.5), and frequency-dependent viscoelastic behavior wasobserved. Consistent with measurements at constant frequency, when thesemeasurements were performed after various intervals of UV irradiation(20-240 minutes, FIG. 9C), both storage and loss moduli increased.Interestingly, the crossover frequency at 7 rad/s was nearly independentof irradiation time. Consistent with these oscillatory data, the gelsrelaxed strain-induced stress on the order of seconds (see SI).

The crossover frequency of a dynamic frequency sweep measurement isoften correlated to the molecular processes underlying crosslinkexchange. In this case, the stress-relaxing process was assigned to behydrolysis of the boronic ester. The rheological data demonstrated thatthrough photocontrolled dynamic covalent crosslinks, an externalstimulus could reversibly alter the spatial structure of a viscoelasticnetwork (crosslink density) without significantly altering the temporalhierarchy (relaxation modes). Therefore, it is presumed that for thisparticular boronic acid/diol combination, the change in equilibriumconstants for E versus Z azobenzene boronic acid, rather than changes inhydrolysis rates, underlay the phototunable change in stiffness.Strategic modifications of the boronic acid and diol structures couldadditionally enable turning of relaxation modes.

Visible-Light Photoswitching

Next, the system was optimized such that photoreversible viscoelasticitycould be achieved with visible light. In addition to lower reactivity,visible light offers enhanced hydrogel penetration. Polymer P2-F₂ (FIG.7) was synthesized. Hydrogels prepared from mixtures of P1 and P2-F₂ at(1:1, 10 w/v % in PBS, pH 7.5) demonstrated reversible sol to geltransitions by alternative irradiation with green (530 nm) and blue (470nm) LEDs. Rheological characterization confirmed that the stiffness ofthe gels could be reversibly controlled, and the hydrogels wereviscoelastic (FIG. 10A). Importantly, gels synthesized from P2-F₂exhibited moduli that were over two factors larger than those formedfrom P2, which may be due to a higher binding affinity of thefluorinated azobenzene boronic acid with diols. Notably, while G′increased as a function of irradiation time, the stress relation rate ofthe gel did not change (FIG. 10B). Thus, this viscoelastic hydrogeldemonstrated stiffness tunability independent of stress relaxation.

Gratifyingly, the P1/P2-F₂ gel (10 w/v % in PBS, pH 7.5) wassufficiently stiff to form freestanding shapes, so the robustness of thegel could be evaluated. Once cut, these hydrogels were able to heal inminutes at room temperature, which was attributed to the dynamicexchange between boronic acids and diols. The gels formed from 1 hour ofirradiation with green light and the thermal half-life of the Zconformation was at least one month; the stiffness being maintained byexcluding blue light from the room's fluorescent lights with filters.Attempts to swell the gels in solutions of PBS were consistent with alightly crosslinked dynamic network: the material was fully dissolvedafter 6 hours at 23 ° C. The long-term utility of these hydrogels can befurther improved by increasing branch functionality and tuning theidentity of the diol end-groups to increase K_(eq).

Cytocompatibility of the gels. To evaluate the cytocompatibility of theP1/P2-F₂ gels, which can be tuned solely with visible light, HeLa cellswere encapsulated in P1/P2-F₂ (10 wt % Dulbecco's Modified Eagle Medium(DMEM) with 10% fetal bovine serum (FBS)), which formed a soft gel. As acontrol, the cytotoxic compound auranofin (20 μM) was introduced intothe gels with encapsulated cells. After 24 hours, the viability of theencapsulated cells was quantified using a rezasurin reduction assay witha fluorescence plate reader. (Uzarski, J. S. et al., Biomaterials 2017,129, 163-175.) Cells encapsulated in the gels maintained >80% viabilityrelative to cells in FBS-modified DMEM cultured on standard plasticsubstrates, while <10% of the control cells exposed to the cytotoxiccompound survived.

Supporting Information General Information

General procedures. Unless otherwise noted, reactions were performedunder Ar atmosphere in oven-dried (120° C.) glassware. Reaction progresswas monitored by thin layer chromatography (Merk silica gel 60 F₂₅₄plates), and visualizing was performed with fluorescence quenching,KMnO₄, or ninhydrin stains. Automated column chromatography wasperformed using SiliCycle SiliaFlash F60 (40-63 μm, 60 Å) in SNAPcartridges on a Biotage Isolera One. Organic solvents were removed invacuo using a rotary evaporator (Büchi Rotovapor R-100, ˜20-200 torr),and residual solvent was removed under high vacuum (<100 mtorr).Water-soluble polymers were purified by dialysis using SnakeSkinDialysis Tubing (3.5 kDa cutoff, 16 mm diameter) purchased from Fisher.

Materials. Commercial reagents were purchased from Sigma-Aldrich, Acros,Alfa Aesar, TCI, or Oakwood and used as received. 4-Arm PEG-NH₂ HCl salt(M_(w) 5 kDa) was purchased from JenKem, and was azeotroped with toluene(3×) and melted under high vacuum (<100 mtorr) prior tofunctionalization.

Instrumentation. Proton nuclear magnetic resonance (¹H NMR) spectra andcarbon nuclear magnetic resonance (¹³C NMR) spectra were recorded onBruker AVANCE-500 spectrometers at 500 MHz and 125 MHz, and referencedto the solvent residual peaks. Boron nuclear magnetic resonance (¹¹BNMR) spectra were recorded on Bruker AVANCE-400 spectrometers at 128MHz. BF₃.OEt₂ in CDCl₃ in a capillary was used as a reference for ¹¹BNMR (0 ppm) in Wilmad Precision NMR tubes (CFQ, 500 MHz, OD: 5 mm, wallthickness: 0.38 mm). NMR data are represented as follows: chemical shift(δ ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet), coupling constant in Hertz (Hz), integration. UV-visspectra were collected on a Cary 5000 UV-vis-NIR spectrophotometer withan Hg lamp; cuvettes were 10-mm path length quartz cells (Starna23-Q-10). Liquid chromatography-mass spectrometry (LCMS) kineticsexperiments were performed on a liquid chromatography-mass spectrometrysystem (Agilent 6120 Quadrupole LC/MS) equipped with avariable-wavelength detector. Infrared spectroscopy was performed on aThermo Nicolet iS10 with a ZnSe crystal ATR attachment. Size exclusionchromatography (SEC) measurements were performed in stabilized,HPLC-grade tetrahydrofuran using an Agilent 1260 Infinity II system withvariable-wavelength diode array (254, 450, and 530 nm) and refractiveindex detectors, guard column (Agilent PLgel; 5 μm; 50×7.5 mm), andthree analytical columns (Agilent PLgel; 5 μm; 300×7.5 mm; 10⁵, 10⁴, and10³ Å pore sizes). The instrument was calibrated with narrow-dispersitypolystyrene standards between 640 Da and 2300 kDa (Polymer StandardsService GmbH). All runs were performed at 1.0 mL/min flow rate and 40°C. Molecular weight values were calculated based on the refractive indexsignal.

Synthesis of Azobenzene Boronic Acids

The synthetic route of small molecules 1 and 2 for kinetic studies andcompound SI-2 and SI-5 for synthesis of control polymers P2 and P3 isshown in Scheme S1 in FIG. 14.

Methyl 4-nitrosobenzoate (SI-1)

Based on a modified from literature procedure, methyl 4-aminobenzoate (3g, 20 mmol) was dissolved in 66 mL of dichloromethane (DCM) into a500-mL round-bottomed flask (RBF) equipped with stir bar. (Priewisch, B.et al., The Journal of Organic Chemistry. 2005, 70, 2350.) Oxone (20 g,40 mmol) was dissolved into 250 mL of deionized water and then added tothe reaction mixture (capped with rubber septum which was pierced with aneedle, exposed to air), and the reaction was aged for 1 hour with highstirring. Over the course of the reaction, the biphasic solutiondeveloped into an intense neon green color. The product was extractedagainst water (1×), 1M HCl (2×), and 1M NaOH (3×). The organic layer wasdried over sodium sulfate and concentrated in vacuo to give methyl4-nitrosobenzoate (2.26 g, 70%). This product was used in the subsequentstep without further purification. ¹H NMR (500 MHz, Chloroform-d) δ 8.30(d, J=8.5 Hz, 2H), 7.94 (d, J=8.5 Hz, 2H), 3.98 (s, 3H).

Methyl(E)-4-((2-(4,4,5,5-tetramethyl-1,3,2,-dioxaborolan-2-yl)phenyl)diazinyl)benzoate(2)

SI-1(4.4 g, 27 mmol) and2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (6.1 g, 28 mmol)were dissolved in 100 mL of DCM into a 250 mL RBF, to which 20 mL ofacetic acid was added. The reaction was stirred for 24 hours, open toair. The solution turned black after several hours. Upon consumption ofthe starting material, the organic layer was washed with 1M HCl (3×),and the organic layer was collected, dried with sodium sulfate, andconcentrated in vacuo. Residual acetic acid was removed under highvacuum. The collected solids were diluted in hexane (300 mL), and theprecipitate which formed (byproduct) was removed with a glass frit. Thered filtrate was concentrated in vacuo to approximately 100 mL and thenfiltered again. The collected filtrate was concentrated in vacuo toyield an orange solid (˜6 g) which was recrystallized from hexane (30mL) twice to provide the product as a red crystalline solid (5 g, 50%).¹H NMR (500 MHz, CDCl₃) δ 8.19 (d, J=8.7 Hz, 2H), 7.94 (d, J=8.6 Hz,2H), 7.81 (d, J=8.0, 1.1 Hz, 1H), 7.75 (d, J=1.5 Hz, 1H), 7.56-7.52 (m,1H), 7.50-7.46 (m, 1H), 3.96 (s, 3H), 1.36 (s, 12H). ¹³C NMR (125 MHz,CDCl₃) δ 166.6, 156.9, 155.4, 134.5, 131.5, 130.8, 130.6, 130.5, 122.8,119.7, 84.0, 52.3, 25.0. ¹¹B NMR (128 MHz, CDCl₃) 31.3. IR (cm⁻¹) 2977,2953, 2925, 2851, 1721, 1597. HRMS m/z expected for C₂₀H₂₄BN₂O₄ [M+H]⁺367.18, measured 367.18.

(E)-(2-((4-(methoxycarbonyl)phenyl)diazinyl)phenyl)boronic acid (1)

2 (5 g, 10 mmol) was dissolved into a 500 mL RBF equipped with a stirbar (capped with a rubber septum which was pierced with a needle,exposed to air). The starting material was dissolved in tetrahydrofuran(THF) (250 mL), providing a clear, deep red solution. Sodium periodate(9 g, 40 mmol) was dissolved in deionized (DI) water (62 mL) andtransferred to the solution. After stirring for 30 minutes, 1M HCl (8mL) was added. The reaction was stirred overnight at room temperature.In the morning, the solution was red and cloudy with precipitate. TheTHF was removed in vacuo to concentrate the product in water. Theresulting suspension was diluted with additional water and filtered toprovide an orange/peach-colored solid. The solids were washed withacetonitrile (100 mL) and collected and dried on a glass filter to givethe 1 (3.5 g, 90%) as a peach-colored solid. This product was used inthe subsequent step without further purification. ¹H NMR (500 MHz,DMSO-d₆) δ 8.19 (d, J=8.4 Hz, 2H), 7.96 (d, J=8.4 Hz, 2H), 7.92 (d, J6.3 Hz, 1H), 7.83 (s, 2H), 7.59-7.53 (m, 3H), 3.91 (s, 3H). ¹³C NMR (126MHz, DMSO-d₆) δ 166.1, 155.0, 154.1, 133.1, 131.9, 131.8, 131.0, 129.6,123.6, 123.0, 52.9. ¹¹B NMR (128 MHz, DMSO-d₆) 28.4. IR (cm⁻¹) 3347(br), 3198 (br), 3033, 2962, 1723, 1597. LRMS m/z expected forC₁₄H₁₄BN₂O₄ [M+H]⁺ 285.10, measured 285.1.

(E)-4-((2-boronophenyl)diazenyl)benzoic acid (SI-2)

1 (3 g, 10 mmol) was dissolved in MeOH (282 mL) in a 1-L RBF equippedwith a stir bar, providing a bright red solution. Lithium hydroxide (1g, 40 mmol) was dissolved into water (58 mL) and transferred to thereaction mixture, which was stirred open to air overnight. The methanolwas removed in vacuo, providing a red alkaline aqueous solution. Thissolution was neutralized with 1M HCl to precipitate the desired product,which was collected on a glass filter and dried in a vacuum oven (100°C.) for 1 hr to provide the title product (2.45 g, 91%) as apeach-colored solid. This product was used in the subsequent stepwithout further purification. ¹H NMR (500 MHz, DMSO-d₆) δ 13.25 (s, 1H),8.17 (m, 2H), 7.98-7.90 (m, 3H), 7.82 (s, 2H), 7.62-7.52 (m, 3H). BC NMR(125 MHz, DMSO-d₆) δ 167.2, 155.1, 154.0, 133.2, 133.1, 131.7, 131.1,129.5, 123.5, 122.9. ¹¹B NMR (128 MHz, DMSO-d₆) 28.6. IR(cm⁻¹) 3395(br), 3106, 3064, 2359, 2339, 2161, 1682, 1601. LRMS m/z expected forC₁₃H₁₀BN₂O₄ [M+H]⁻ 269.07, measured 269.1.

(E)-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)diazenyl)benzoate(SI-3)

SI-1(1.1 g, 6.60 mmol) and4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1.6 g, 7.3 mmol)were dissolved in DCM (100 mL) in a 200 mL RBF. Acetic acid (5 mL) wasadded dropwise. Initially the solution was neon green, but over thecourse of 24 hours the reaction turned dark red. Upon completion, thereaction was extracted against water (1×), 1M HCl (1×), and brine (1×).The combined organics were concentrated in vacuo and dissolved in DCM,which was passed through a silica plug and concentrated in vacuo. Theresulting product was recrystallized from 9:1 hexane:EtOAc to providethe title product (712 mg, 30%). ¹H NMR (500 MHz, CDCl₃) δ 8.20 (d,J=8.6 Hz, 2H), 8.01-7.95 (m, 4H), 7.93 (d, J=8.3 Hz, 2H), 3.96 (s, 3H),1.38 (s, 12H). ¹³C NMR (125 MHz, CDCl₃) δ 166.5, 155.2, 154.2, 135.7,131.9, 130.6, 122.7, 122.2, 84.2, 52.4, 24.9. ¹¹B NMR (128 MHz, CDCl₃)30.1. IR(cm⁻¹) 2978, 2956, 2928, 2361, 2343, 1716, 1601. HRMS: m/zexpected for C₂₀H₂₄BN₂O₄ [M+H]⁺ 367.18, measured 367.18.

(E)-(4-((4-(methoxycarbonyl)phenyl)diazenyl)phenyl)boronic acid (SI-4)

SI-3 (510 mg, 1.39 mmol) was dissolved in 25 mL of THF into a 100 mL RBFequipped with a stir bar to give a red-colored solution. Sodiumperiodate (298 mg, 1.39 mmol) was dissolved in water (6.2 mL) and addedto the reaction. After 30 minutes, 1 mL of 1 M HCl was added to thesolution, which was stirred for a further 6 hours. Upon full conversion,the THF was removed in vacuo, leading to the precipitation of theproduct, which was collected on a glass frit. The solids were washedwith water (3×10 mL) and acetonitrile (10 mL) and dried to provide thetitle product (320 mg, 81%) as a peach-colored solid. ¹H NMR (500 MHz,DMSO-d₆) δ 8.30 (s, 2H), 8.18 (d, J=8.2 Hz, 2H), 8.05-7.99 (m, 4H), 7.90(d, J=7.9 Hz, 2H), 3.92 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 166.1,154.5.0, 153.4, 135.7, 132.1, 131.0, 123.2, 122.2, 52.9. ¹¹B NMR (128MHz, DMSO-d₆) 26.8. IR (cm⁻¹) 3346 (br), 2965, 2358, 1724, 1604. HRMSm/z expected for C₁₄H₁₄BN₂O₄ [M+H]⁺ 285.10, measured 285.10.

(E)-4-((4-boronophenyl)diazenyl)benzoic acid (SI-5)

SI-4 (1.19 g, 4.19 mmol) was dissolved in methanol (112 mL) in a 250-mLRBF equipped with a stir bar. Lithium hydroxide (0.401 g, 16.8 mmol) wasdissolved in water (23 mL) and added to the solution, which was stirredovernight. After 14 hours, the reaction was as an orange solution. Themethanol was removed in vacuo and then 1M NaOH was added to the crudemixture to dissolve the solids. The aqueous solution was washed withEtOAc and then neutralized with 1M HCl, leading to the precipitation ofthe product. The solids were filtered, washed with water, and dried toprovide the title product (160 mg, 50%) as a peach-colored solid. ¹H NMR(500 MHz, DMSO-d₆) δ 8.10 (d, J=8.2 Hz, 2H), 7.99 (d, J=7.9 Hz, 2H),7.89 (d, J=8.1 Hz, 2H), 7.86 (d, J=8.1 Hz, 2H). ¹³C NMR (125 MHz,DMSO-d₆) δ 168.7, 1538, 153.5, 138.3, 135.6, 130.8, 122.6, 122.0. ¹¹BNMR (128 MHz, DMSO-d₆) 26.5. IR (cm⁻¹) 3406 (br), 2359, 1692, 1601. HRMSm/z expected for C₁₃H₁₀BN₂O₄ [M+H]⁻ 269.07, measured 269.07.

4-amino-3,5-difluorobenzoic acid

4-Amino-3,5-difluorobenzoic acid was synthesized by following apreviously reported procedure. Characterization data were consistentwith literature reports. (Bléger, D. et al., J. Am. Chem. Soc. 2012,134, 20597.)

Methyl 4-amino-3,5-difluorobenzoate (SI-6)

4-Amino-3,5-difluorobenzoic acid (500 mg, 2.89 mmol) was dissolved in 20mL of MeOH, to which 1.75 mL of concentrated sulfuric acid was added.The reaction was heated to reflux for 14 hours. After the reaction, thesolvent was removed in vacuo and 20 mL of water was added, leading tothe precipitation of the product, which was collected and dried on afunnel to provide the title product (417 mg, 77%) as a white solid,which was used in the next reaction without further purification. ¹H NMR(500 MHz, CDCl₃) δ 7.58-7.46 (m, 2H), 4.14 (s, 2H), 3.87 (s, 3H).

Methyl 3,5-difluoro-4-nitrosobenzoate (SI-7)

Methyl 4-amino-3,5-difluorobenzoate (2.52 g, 13.5 mmol) was dissolved inDCM (60 mL) into a 500 mL RBF equipped with a stir bar, and oxone (30.2g, 49.1 mmol) in water (240 mL) was added to the solution, which wasleft to react for 24 hours. The organic layer was washed with 1M HCl(1×), 1M NaOH (1×), and DI water (1×). This was concentrated to give thetitle product (2.05 g, 76%) as a yellow solid, which was used in thenext reaction without further purification.

(E)-(2-((2,6-difluoro-4-(methoxycarbonyl)phenyl)diazenyl)phenyl)boronicacid (SI-8)

SI-7 and 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (2.23 g,10.2 mmol) were weighed into a 100 mL RBF equipped with a stir rod underinert atmosphere. DCM (70 mL) was added, and the dissolution of bothstarting materials led to the development of a dark-colored solution.Acetic acid (12 mL) was added, and the solution was left to react for 24hours. After 24 hours, the solvent was removed in vacuo and with highvacuum (to remove excess acetic acid), and the crude residue was dilutedin 100 mL of hexane and filtered to provide the protected crude (2.17 g)as a red solid. This material was dissolved in 130 mL of THF into a 250mL RBF equipped with a stir rod. Sodium periodate (3.46 g, 16.2 mmol)was added in 30 mL of water, and the solution was left to react for 14hours. After the reaction, the THF was removed in vacuo, and theremaining crude was diluted with 100 mL of DCM. The product precipitatedfrom solution and was collected on a Buchner funnel, which was washedwith an additional 100 mL of DCM to yield the title product (1.12 g,34%) as a dark orange solid. ¹H NMR (500 MHz, DMSO-d₆) δ 7.99 (s, 2H),7.87-7.80 (m, 3H), 7.71-7.67 (m, 1H), 7.63-7.57 (m, 2H), 3.92 (s, 3H).¹³C NMR (125 MHz, DMSO-d₆) δ 164.2, 156.1, 156.0, 154.1, 134.2, 132.9,130.2, 120.5, 114.4, 114.2, 53.5 ¹⁹F NMR (#MHz, DMSO-d₆) δ -116.6 (E),−119.8 (Z) IR (cm⁻¹) 3266 (br), 2361, 1724, 1592. LRMS m/z expected forC₁₄H₁₁BF₂N₂O₄ [M+H]⁺ 321.08, measured 321.1.

(E)-4-((2-boronophenyl)diazenyl)-3,5-difluorobenzoic acid (SI-9)

SI-8 (70 mg, 0.22 mmol) was dissolved in 7 mL of MeOH into a 25 mL RBFequipped with a stir bar, to which LiOH (16 mg, 0.66 mmol) in 3 mL ofwater was added. The reaction was left to react for 14 hours. After thereaction, the methanol was removed in vacuo and the water wasneutralized with 1M HCl, leading to the precipitation of the product.The product was dissolved in EtOAc and washed with water. The organiclayer was dried with sodium sulfate and then concentrated in vacuo togive the title product (64.4 mg, 96%) as an orange solid. ¹H NMR (500MHz, DMSO-d₆) δ 7.99 (s, 2H), 7.83 (d, J=7.4 Hz, 1H), 7.79 (d, J=9.5 Hz,2H), 7.69 (s, 1H), 7.60 (tt, J=7.3, 5.6 Hz, 2H) ¹³C NMR (125 MHz,DMSO-d₆) δ 165.2, 156.1, 156.0, 154.1, 134.2, 132.8, 130.2, 120.2,114.3, 114.1 ¹⁹F NMR (#MHz, DMSO-d₆) δ -117.0 (E), −120.1 (Z) IR (cm⁻¹)3361 (br), 3053, 2358, 1720, 1595. LRMS m/z expected for C₁₃H₉BF₂N₂O₄[M+H]⁺ 306.06, measured 307.0.

Small-Molecule Kinetics Procedure for Calibration Curves

Stock solutions of (E)-1 or (E)-2 were prepared in acetonitrile at 800μM. Serial dilutions were performed to obtain a final concentration of12.5 μM. The solutions were directly injected onto the LCMS (Poroshell120 column, EC-C18, 2.7 μm, 2.1×50 mm). Runs were ten minutes long withsolvent gradient from 50:50 to 100:0 ACN:H₂O in the first three minutes.Detection was performed at 254 nm with a variable-wavelength detector.The peak area for each concentration was recorded. A calibration curvewas created. The Z-isomer calibration curves were prepared byirradiating the series above for 10 minutes with 365 nm UV light anddirectly injecting the solution onto the LCMS. The concentration of theZ-isomer can be calculated from the known concentration of the E-isomer(based on the previously-generated calibration curve).

Pseudo-First Order Reversible Esterification of (E)-1

500 μL of a stock solution of 800 μM of (E)-1 was diluted with 500 μL of80 mM of pinacol in DI water. The concentrations of (E)-1 and (E)-2 weremonitored as a function of time using the above LCMS method. Theobserved rate constants of esterification (k_(1(E))) and hydrolysis(k_(−1(E))) were determined using the derivation shown below.

Scheme S2. Chemical structures for the pseudo-first order reversibleesterification of (E)-1 and/or (Z)-1. The photochemical E/Zisomerization is drawn as irreversible due to the slow thermalrelaxation of (Z)-1 and (Z)-2. This also allows for the simplificationof the derivation of observed rate constants, shown below.

The data from the concentrations vs. time experiments was fit to thelinear expression (1) for a reversible first order reaction and yieldsthe slope denoted as equation (4). (Bléger, D. et al., 2012.) Theapparent equilibrium constant for the reaction is the ratio of (E)-2 to(E)-1 when the reaction comes to equilibrium, which can also berepresented as equation (2) and rewritten as equation (3). With theapparent equilibrium constant and slope known, the rates of hydrolysiscan be determined with equation (5), and subsequently the rate ofesterification can be determined.

$\begin{matrix}{{{{\ln\left( {\left\lbrack {(E) - 2} \right\rbrack_{e} - {\ln\left\lbrack {(E) - 2} \right\rbrack}_{t}} \right)} - {\ln\left( {\left\lbrack {(E) - 2} \right\rbrack_{e} - {\ln\left\lbrack {(E) - 2} \right\rbrack}_{0}} \right)}} = {\left( {k_{1{(E)}}^{\prime} - k_{{- 1}{(E)}}^{\prime}} \right)t}}{{{where}k_{{- 1}{(E)}}^{\prime}} = {{{k_{{- 1}{(E)}}\left\lbrack {H_{2}O} \right\rbrack}{and}k_{1{(E)}}^{\prime}} = {k_{1{(E)}}\left\lbrack {(E) - 1} \right\rbrack}}}} & (1)\end{matrix}$ $\begin{matrix}{K_{{eq}(E)}^{\prime} = \frac{k_{1{(E)}}^{\prime}}{k_{{- 1}{(E)}}^{\prime}}} & (2)\end{matrix}$ $\begin{matrix}{{k_{{- 1}{(E)}}^{\prime}K_{{eq}(E)}^{\prime}} = k_{1{(E)}}^{\prime}} & (3)\end{matrix}$ $\begin{matrix}{{k_{{- 1}{(E)}}^{\prime} + k_{1{(E)}}^{\prime}} = {slope}} & (4)\end{matrix}$ $\begin{matrix}{k_{{- 1}{(E)}}^{\prime} = \frac{slope}{1 + K_{{eq}(E)}^{\prime}}} & (5)\end{matrix}$

Pseudo-First Order Reversible Esterification of (Z)-1 and (E)-1

500 μL of a stock solution of 800 μM of (E)-1 was irradiated with a 365nm flashlight for 10 minutes to produce a mixture of (E)-1 and (Z)-1.The mixture was then diluted with 500 μL of a 80 mM pinacol solution inDI water, and the concentrations of (E)-2 and (Z)-2 were monitored as afunction of time using the above LCMS method. The observed rateconstants of esterification and hydrolysis k_(1(Z)) and k_(−1(Z)) weresolved for using the same derivation as shown for the E isomer. A smallincrease in the total concentrations of (E)-1 and (E)-2 over the courseof this experiment were ascribed to thermal relaxation of the (Z)isomers.

Pseudo-First Order Irreversible Hydrolysis of (Z)-2 and (E)-2

500 μL of a stock solution of 800 μM of (E)-2 was irradiated with a 365nm flashlight for 10 minutes to produce a mixture of (E)-2 and (Z)-2.The mixture was then diluted with 500 μL of DI H₂O and the concentrationof (E)-2 and (Z)-2 was monitored as a function of time. The observedrate constants of hydrolysis k_(−1(E)) and k_(−1(Z)) were determinedusing the derivation shown below.

Scheme S3. Chemical structures for the pseudo-first order irreversiblehydrolysis of (E)-2 and/or (Z)-2. The photochemical E/Z isomerization isdrawn as irreversible due to the slow thermal relaxation of (Z)-1 and(Z)-2. This also allows for the simplification of the derivation ofobserved rate constants, shown below.

The concentrations vs. time can be fit to the linear expression (6) foran irreversible first order reaction where the slope is the apparentrate constant for the respective isomer. (Hartley, F. R., Chem. Br.1984, 20, 148.)

ln[(E)-2]_(t)−ln[(E)-2]₀ =−k′ _(−1(E)) t  (6)

where k′_(−1(E))=k_(−1(E))[H₂O]

Functionalization of 4-Arm PEG

P1 was prepared following a literature procedure. (Yesilyurt, V. et al.,Adv. Mater. 2016, 28, 86.)

P2 was synthesized based on an adapted literature procedure. (Yesilyurt,V. et al, Adv. Mater. 2017, 29.) 500 mg of 4-arm PEG amine HCl salt(M_(w) 5000) was added to a Schlenk flask (50 mL) and melted under highvacuum (3×) to remove excess water. HOBt (0.2 g, 1 mmol),benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(0.4 g, 1 mmol), and SI-2 (0.3 g, 1 mmol) were then added as solidsalong with a stir bar. The vessel was sealed and backfilled withnitrogen three times. 5 mL of DCM and 5 mL of dimethylformamide (DMF)were then added to solubilize the reagents, leading to a clear redsolution. Triethylamine (91 mg, 125 μL, 0.90 mmol) was added to thesolution, which was stirred at room temperature for 24 hours. Thesolvent was removed in vacuo, and the solution was diluted with DI waterto form an orange precipitate. The solids were removed by filtrationusing a fritted funnel, and the aqueous orange filtrate was subject todialysis (MWCO=3.5 kDa) against DI water for 24 hours, during which timethe dialysate was changed at least 3 times. After dialysis, the samplewas lyophilized for 48 hours, yielding 320 mg of an orange powder, P2.Polymers should be stored dry in fridge without exposure to light topreserve integrity. Leaving solutions of P2 in DI water in ambient lightfor 24 hours leads to protodeboronation, as evinced by proton NMR.

P2-F₂ was synthesized using the same procedure as P2, using SI-10. Afterdialysis, this solution was concentrated and purified using a spinfilter (MWCO=5 KDa). After the reaction, it was lyophilized for 48 hours(91 mg yield).

P3 was synthesized by the same procedure as P2, using SI-5 (377 mgyield).

P4 was synthesized by same procedure as P2, using(E)-4-(phenyldiazenyl)benzoic acid (217 mg yield).

Rheology

Mechanical characterization of the prepared hydrogels was performedusing an Anton Paar MCR 302 Rheometer with a 25 mm, 5° cone-plateattachment. 10% strain was established to be within the linearviscoelastic regime for all time points tested. Unless noted otherwise,oscillatory strain amplitude sweeps were conducted using a frequency of25 rad/s, and oscillatory frequencies were conducted using 10% strain.Gelation profiles were conducted with 10% strain and a frequency of 25rad/s. Frequency sweeps were performed at 10% strain, with a frequencyrange of 300 to 1 rad/s. Data were collected at 25° C. Gels wereprepared by mixing 2004 of P1 (10 w/v % in 0.1M PBS, pH 7.5) with P2,P3, or P4 (10 w/v % in 0.1M PBS, pH 7.5).

A consistent observation from the reversible gelation profiles is thatthe first stiffening event occurred approximately twice as slowlycompared to subsequent cycles. This may be due to (a) a slightlyelevated temperature of the glass plate of the photorheology setup aftercontinuous irradiation, (b) diffusion processes in the macroscopic gel,or (c) the photostationary state after blue irradiation was <100% Eisomer. While it was not possible to control this temperature apart fromimplementation of a fan, no more than a 3° C. increase in temperaturewas observed at the sample as measured by as measure by a fluke 62 maxIR thermometer.

TABLE 2 Tabulated data of low frequency crossover points and respectivemodulus (Pa) of gels prepared from P1 and P2 (1:1, 10 w/v % in 0.1M PBS,pH 7.5, 10% strain) at different irradiation intervals. Time Irradiatedat 365 nm ω_(c) G at ω_(c) 20 7.49 5.94 40 7.98 11 60 8.1 17.5 90 7.7832.2 120 7.45 47.5 180 7.11 84.3 240 7.75 141

Example 2: Alginate-Based Hydrogels

A ring opening reaction between δ-gluconolactone and sodium alginate, apolysaccharide commonly used in biomaterials, was used to form awater-soluble, unhindered penta-ol-grafted polymer (FIG. 11A). Thegrafted alginate formed photoresponsive gels in combination with atelechelic bis-azobenzene boronic acid having ortho-fluorine atoms onthe arylboronic acid ring as a linker.

The alginate-based gels exhibited reversibly photocontrolled stiffness(with a maximum stiffness of 9 kPa (FIG. 11B). The alginate-based gelsdisplayed significantly slower stress relaxation than PEG-based gels(FIG. 11C). However, as in the PEG gels, the stress relaxation ofalginate gels was constant as a function of irradiation and stiffness.

Example 3: Hybrid Collagen/Poly(ethylene glycol)-Based InterpenetratingNetwork

5 mg of P1 (10 kDa-8 arm) and 5 mg P2-F₂ (5 kDa, 4-arm) were added to asolution of 20 μL of 10×PBS, 2.8 μL of 1M NaOH, 57.2 μL of deionizedwater, and 48 μL of 20 mM acetic acid. 72 μL of stock collagen solutionwas added to this and fibrillogensis was set in the solution state byincubating the gel for 30 minutes at 37° C. Rheological data wascollected at 10% strain and 25 rad/s.

Example 4: Hybrid ECM/Poly(ethylene glycol)-Based InterpenetratingNetwork

A 20 w/v % stock solution of P2-F₂-5kDa was prepared in deionized waterand a 20 w/v % stock solution of P1-5 kDa was prepared in 2×PBS buffer(pH 7.4). 50 μL of P1 solution and 50 μL of P2-F₂ solution were combinedwith 100 μL of 10×PBS pH (7.5 s) and then added to 100 μL decellularizedrat liver ECM (5 w/v %, neutralized) that had been gelled for 30 min at37° C. Rheological data were collected at 10% strain and 25 rad/s.

The word “illustrative” is used herein to mean serving as an example,instance, or illustration. Any aspect or design described herein as“illustrative” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Further, for the purposes ofthis disclosure and unless otherwise specified, “a” or “an” means “oneor more.”

The foregoing description of illustrative embodiments of the inventionhas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of theinvention. The embodiments were chosen and described in order to explainthe principles of the invention and as practical applications of theinvention to enable one skilled in the art to utilize the invention invarious embodiments and with various modifications as suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and theirequivalents.

What is claimed is:
 1. A reversibly crosslinkable compositioncomprising: a first molecule comprising at least two terminal azobenzeneboronic acid groups; and a second molecule comprising at least twoterminal diol groups, wherein at least one of the first and secondmolecules is a polymer, and further wherein, if the first molecule hasonly two terminal azobenzene boronic acid groups, then the secondmolecule comprises at least three terminal diol groups, and if thesecond molecule has only two terminal diol groups, the first moleculehas at least three terminal azobenzene boronic acid groups; wherein thefirst molecule is characterized in that its azobenzene groups undergo areversible E to Z isomerization upon irradiation with ultraviolet light,visible light, or infrared light, and its boronic acid groups react withthe terminal diol groups of the second polymer to form a polymer networkcomprising covalent azobenzene boronic ester bonds.
 2. The compositionof claim 1, wherein one or both of the first molecule and the secondmolecule comprise polysaccharide chains.
 3. The composition of claim 2,wherein the polysaccharide chains comprise hyaluronic acid chains. 4.The composition of claim 1, wherein one or both of the first moleculeand the second molecule comprise poly(ethylene glycol) chains.
 5. Thecomposition of claim 1, wherein one or both of the first molecule andthe second molecule comprise decellularized extracellular matrix.
 6. Thecomposition of claim 1, wherein one or both of the first molecule andthe second molecule comprise fiber-forming components of extracellularmatrix.
 7. The composition of claim 1, wherein one or both of the firstmolecule and the second molecule comprise alginate chains.
 8. Thecomposition of claim 1, wherein the azobenzene groups areortho-difluoroazobenzene groups.
 9. The composition of claim 1, whereinat the azobenzene groups comprise an electron withdrawing group para tothe boronic ester group.
 10. A method of inducing a reversible polymernetwork formation in a reversibly crosslinkable composition comprising:a first molecule comprising at least two terminal azobenzene boronicacid groups; and a second molecule comprising at least two terminal diolgroups, wherein at least one of the first and second molecules is apolymer, and further wherein, if the first molecule has only twoterminal azobenzene boronic acid groups, then the second moleculecomprises at least three terminal diol groups, and if the secondmolecule has only two terminal diol groups, the first molecule has atleast three terminal azobenzene boronic acid groups; wherein the firstmolecule is characterized in that its azobenzene groups undergo areversible E to Z isomerization upon irradiation with ultraviolet light,visible light, or infrared light, and its boronic acid groups react withthe terminal diol groups of the second polymer to form a polymer networkcomprising covalent azobenzene boronic ester bonds, the methodcomprising: irradiating at least a portion of the composition withultraviolet light, visible light, or infrared light that induces theazobenzene groups of the first molecule to undergo a reversible E to Zisomerization, whereby its boronic acid groups react with the terminaldiol groups of the second molecule to form a polymer network comprisingcovalent azobenzene boronic ester bonds.
 11. The method of claim 10,further comprising removing one or more portions of the composition thatwere not irradiated by the ultraviolet light, visible light, or infraredlight.
 12. The method of claim 10, further comprising irradiating atleast a portion of the polymer network with visible light that inducesthe azobenzene groups of the first polymer to undergo a reversible Z toE isomerization, whereby azobenzene boronic ester bonds in the polymernetwork are broken.
 13. The method of claim 10, wherein two or moredifferent regions of the composition are irradiated with the ultravioletlight, visible light, or infrared light for different time durations, atdifferent light intensities, or at different wavelengths of light, suchthat the two or more different regions of the polymer network havedifferent stiffness properties.
 14. The method of claim 10, wherein afirst region of the two or more different regions of the composition hasa G′_(max) that is at least twice the G′_(max) of a second of the two ormore different regions of the composition.
 15. The method of claim 10,further comprising seeding the polymer network with living biologicalcells.
 16. The method of claim 15, further comprising culturing thebiological cell-seeded polymer network in a cell growth culture medium.17. The method of claim 10, wherein the polymer network comprisespolysaccharide chains.
 18. The method of claim 17, wherein thepolysaccharide chains comprise hyaluronic acid chains.
 19. The method ofclaim 10, wherein the polymer network comprises poly(ethylene glycol)chains.